Recombinant Anti-VAV3 antibody [EP1130Y] (ab52938)

False colour image of Western blot: Anti-VAV3 antibody [EP1130Y] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52938 was shown to bind specifically to VAV3. A band was observed at 98 kDa in wild-type HeLa cell lysates with no signal observed at this size in VAV3 knockout cell line ab265419 (knockout cell lysate ab258280). To generate this image, wild-type and VAV3 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Immunocytochemistry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling VAV3 with purified ab52938 at 1/50 dilution (7.2 µg/mL). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Purified ab52938 at 1/30 dilution (2µg) immunoprecipitating VAV3 in Jurkat whole cell lysate.
Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10µg.
Lane 2 (+): ab52938 + Jurkat whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52938 in Jurkat whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.

Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labelling VAV3 with purified ab52938 at 1/40 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).